Muscle targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping of exon 44 of dystrophin in a subject

Abstract

Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, e.g., an oligonucleotide that causes exon skipping in a mRNA expressed from a mutant DMD allele.

What is claimed is: 
     
       1. A complex comprising an anti-transferrin receptor antibody covalently linked to an exon-skipping oligonucleotide that induces skipping of exon 44 of dystrophin (DMD),
 wherein the anti-transferrin receptor antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: 
 the VH comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 438, and comprises humanized framework regions, and 
 the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 439, and comprises humanized framework regions; and 
 wherein the oligonucleotide is 20 to 27 nucleotides in length and comprises a region of complementarity to exon 44 of a DMD transcript as set forth in SEQ ID NO: 295, wherein the region of complementarity is at least 12 nucleotides in length, and wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). 
 
     
     
       2. The complex of  claim 1 , wherein the oligonucleotide is 26 nucleotides in length. 
     
     
       3. The complex of  claim 2 , wherein the region of complementarity is at least 16 nucleotides in length. 
     
     
       4. The complex of  claim 3 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 69, 71, 96, and 97. 
     
     
       5. The complex of  claim 3 , wherein the oligonucleotide comprises the sequence of SEQ ID NO: 69 or 71. 
     
     
       6. The complex of  claim 1 , wherein the oligonucleotide is 26 nucleotides in length, comprises the sequence of SEQ ID NO: 69 or 71, and is fully complementary to a cognate sequence of SEQ ID NO: 295. 
     
     
       7. The complex of  claim 4 , wherein the drug to antibody ratio (DAR) of the complex is in a range of 1 to 5. 
     
     
       8. The complex of  claim 4 , wherein the anti-transferrin receptor antibody is covalently linked to the oligonucleotide at the 3′ end of the oligonucleotide. 
     
     
       9. The complex of  claim 1 , wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined according to the Chothia numbering system. 
     
     
       10. The complex of  claim 9 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 69, 71, 96, and 97. 
     
     
       11. The complex of  claim 10 , wherein the drug to antibody ratio (DAR) of the complex is in a range of 1 to 5. 
     
     
       12. The complex of  claim 9 , wherein the anti-transferrin receptor antibody is in the form of a full-length IgG comprising a human IgG1 heavy chain constant region, or a functional variant thereof, and a human kappa light chain constant region. 
     
     
       13. The complex of  claim 9 , wherein the anti-transferrin receptor antibody comprises a human kappa light chain constant region and a human IgG1 heavy chain constant region comprising at least one amino acid substitution relative to a wild-type human IgG1 heavy chain constant region. 
     
     
       14. The complex of  claim 12 , wherein the full-length IgG comprises a heavy chain constant region comprising at least one amino acid substitution that alters the effector function of the anti-transferrin receptor antibody. 
     
     
       15. The complex of  claim 14 , wherein the alteration of the effector function comprises reduced Fc receptor binding. 
     
     
       16. The complex of  claim 14 , wherein the full-length IgG comprises two or more amino acid substitutions in a CH2 domain and two or more amino acid substitutions in a CH3 domain, relative to a full-length IgG comprising an IgG1 constant region having an amino acid sequence of SEQ ID NO: 287. 
     
     
       17. The complex of  claim 12 , wherein one or more amino acid residues in the N-terminal region of a CH2 domain of the antibody are altered. 
     
     
       18. The complex of  claim 13 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules. 
     
     
       19. The complex of  claim 18 , wherein the one or more sugar or carbohydrate molecules comprise a fucose unit. 
     
     
       20. The complex of  claim 1 , wherein the complex is formable by a process comprising reacting a first electrophile of a linker precursor compound and a nucleophile of the anti-transferrin receptor antibody. 
     
     
       21. The complex of  claim 20 , wherein the nucleophile of the anti-transferrin receptor antibody is a thiol group. 
     
     
       22. The complex of  claim 21 , wherein the nucleophile of the anti-transferrin receptor antibody is a thiol group of a cysteine of the anti-transferrin receptor antibody. 
     
     
       23. The complex of  claim 22 , wherein the linker is covalently linked to the anti-transferrin receptor antibody via an amide bond, a hydrazide, a trizaole, a thioether, or a disulfide bond. 
     
     
       24. The complex of  claim 23 , wherein the linker comprises one or more moieties selected from the group consisting of: a valine-citrulline sequence, an alanine-citrulline sequence, maleimide, and an alkyl group. 
     
     
       25. The complex of  claim 20 , wherein the first electrophile of the linker precursor compound is a maleimide moiety, and wherein the nucleophile is a thiol group of a cysteine residue of the anti-transferrin receptor antibody. 
     
     
       26. The complex of  claim 25 , wherein the maleimide moiety is present in a (maleimidomethyl)cyclohexane-1-carboxylate group of the linker precursor compound. 
     
     
       27. The complex of  claim 25 , wherein the complex is formable by a process comprising reacting a second electrophile of the linker precursor compound and a nucleophile covalently attached to the oligonucleotide. 
     
     
       28. The complex of  claim 27 , wherein the second electrophile of the linker precursor compound is a succinimidyl ester. 
     
     
       29. The complex of  claim 28 , wherein the nucleophile covalently attached to the oligonucleotide comprises an alkylamino group. 
     
     
       30. A method of inducing skipping of exon 44 of a DMD pre-mRNA in a muscle cell of a subject expressing a DMD pre-mRNA, the method comprising administering to the subject the complex of  claim 1 , wherein the oligonucleotide promotes the expression or activity of a functional dystrophin protein in a muscle cell of the subject.